The long-term objective of our research is to elucidate the role of DNA methylation in the development and function of the central nervous system (CNS). DNA methylation is a major epigenetic factor involved in gene regulation, genomic imprinting, and X-inactivation. Aberrant DNA methylation has been associated with human diseases including cancer and mental retardation disorders. In the past years, we have studied the role of the maintenance DNA methyltransferase I (Dnmt1) in mouse brain development by generating CNS-specific knockouts. We found that Dnmt1 mutant mice exhibit severe phenotypes including precocious astrogliogenesis, abnormal neuronal maturation, and apoptosis of hypomethylated neural cells during postnatal development. However, our knowledge is still very limited with respect to how DNA methylation is dynamically regulated during CNS development and how aberrant methylation pattern in nerve cells could cause CNS dysfunction leading ultimately to mental retardation disorders. For example, we found that the two de novo DNA methyltransferases, Dnmt3a and Dnmt3b, are differentially expressed in neural precursor cells and postmitotic neurons within specific developmental windows. Furthermore, Dnmt1 and Dnmt3a appeared to co-express in neural precursor cells and postmitotic neurons. We therefore hypothesize that Dnmt1 and Dnmt3a play both overlapping and distinct functions in regulating DNA methylation in neural development and function. In this proposal, we will compare and contrast the phenotypes of CNS-specific conditional mutants that are deficient in Dnmt1, Dnmt3a, or both enzymes. In Aim 1, we will first generate Nestin-cre;Dnmt3a conditional mutant mice and characterize the mutant phenotypes in neuronal and glial differentiation. The phenotype of Nestin- cre;Dnmt3a will be compared with that in Nestin-cre;Dnmt1 conditional mutants. In Aim 2, we will use CamKIIa-cre to generate neuron-specific knockouts of Dnmt1, Dnmt3a or both enzymes in postnatal forebrain. The novel roles for Dnmt3a and/or Dnmt1 in postnatal neuronal survival, morphology, and learning and memory behavior will be studied. In Aim 3, we will use next-generation sequencing technology to decipher genome-wide DNA methylation patterns at single nucleotide resolution. By correlating methylation changes in specific gene promoter regions with altered neuronal gene expression, we shall gain insight into both overlapping and specific roles of Dnmt1 and Dnmt3a in forebrain excitatory neurons. Our proposed project will help us understand the disease mechanisms of neurological disorders caused by the perturbation of DNA methylation activities.